Keratin 8 Mutations are Risk Factors for Developing Liver Disease of Multiple Etiologies

ABSTRACT

Keratin 8 and 18 (K8/K18) mutations are shown to be associated with a predisposition to liver or biliary tract disease, particularly noncryptogenic hepatobiliary disease. Unique K8/K18 mutations are shown in patients with diseases including but without limitation to viral hepatitis, biliary atresia, alcoholic cirrhosis and other acute or chronic toxic liver injury, cryptogenic cirrhosis, acute fulminant hepatitis, autoimmune liver disease, cystic fibrosis, primary biliary cirrhosis, primary sclerosing cholangitis, diseases that are linked with cryptogenic cirrhosis, such as nonalcoholic steatohepatitis, and the like. Livers with keratin mutations had increased incidence of cytoplasmic filamentous deposits. Therefore, K8/K18 are susceptibility genes for developing cryptogenic and noncryptogenic forms of liver disease. Mutant alleles are associated with disease susceptibility, and their detection is used in the diagnosis of a predisposition to these conditions.

SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contracts DK047918& DK007056 awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

INTRODUCTION

Keratin mutations are associated with several skin, oral, esophageal,ocular and cryptogenic liver diseases that reflect tissue-specificexpression of the particular keratin involved. The resulting cellularand tissue defects are manifestations of the clearly defined function ofkeratins that provides cells with the ability to cope with mechanicalstresses. This keratin cytoprotective effect is evident in theblistering phenotype of several human keratin skin diseases such asepidermolysis bullosa simplex, and the phenotypes of animal models thatlack or express a mutant keratin. Also, emerging evidence suggests thatkeratins protect cells from nonmechanical forms of injury via severalmechanisms that may include: keratin regulation of cell signalingcascades, regulation of apoptosis, regulation of the availability ofother cellular proteins, and protein targeting to subcellularcompartments.

The function of keratins in protecting cells from mechanical stress isrelated to their unique properties and abundance as one of three majorcytoskeletal protein families, which include intermediate filaments(IF), microfilaments and microtubules. Keratins (K) are members of theIF protein family, and are specifically expressed in epithelial cellsand their appendages. They consist of >20 members (K1-K20), and arefurther classified into type I (K9-K20) and type II (K1-K8) keratinswhich form obligate, noncovalent heteropolymers. Keratins serve asimportant cell-type-specific markers. For example, unique keratincomplements distinguish different epithelial cell types and therebyreflect epithelial subtype-specific diseases that result fromkeratin-specific mutations. As such, keratinocytes express K5/K14basally and K1/K10 suprabasally, and hepatocytes express K8/K18. K8/K18are also found in other glandular cells including enterocytes, withvariable complements of K19/K20/K7 depending on the cell type.

Most keratin diseases are autosomal-dominant with near completepenetrance. Exceptions appear to be K18 and K8 mutations in patientswith cryptogenic cirrhosis. To date, 6 patients have been described withK8 (5 patients) or K18 (1 patient) mutations, from a group of 55patients with cryptogenic cirrhosis. Most patients with cryptogeniccirrhosis, including those with K8/K18 mutations, do not have awell-defined liver disease family history. Absence of a clear familyhistory suggests that K8/K18 mutations predispose to, rather than cause,liver disease. The presence and frequency of keratin mutations innoncryptogenic liver disease is heretofore unknown.

SUMMARY OF THE INVENTION

Keratin 8 and 18 (K8/K18) mutations are shown to be associated with apredisposition to liver disease, particularly noncryptogenic liverdisease. Unique K8/K18 mutations are shown in patients with diseasesincluding biliary atresia, acute fulminant hepatitis, viral hepatitis Bor C, alcoholic liver disease, primary biliary cirrhosis, autoimmunehepatitis, and the like. Livers with keratin mutations had increasedincidence of cytoplasmic filamentous deposits. Therefore, K8/K18 aresusceptibility genes for developing cryptogenic and noncryptogenic formsof liver disease. Alleles are associated with disease susceptibility,and their detection is used in the diagnosis of a predisposition tothese conditions.

The invention also provides methods for the identification of compoundsthat modulate the expression of genes or the activity or the cellularorganization of gene products involved in liver disease, as well asmethods for the treatment of liver disease, which may involve theadministration of such compounds to individuals exhibiting liver diseasesymptoms or tendencies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B: Protein domains analyzed for K8/K18 mutations and anexample of the identification of a K8 mutation. (A) A central roddomain, consisting of α-helical subdomains, is flanked by non-α-helicalhead/tail domains. The head/tail domains are further subdivided into E,V and H regions. The subdomains of the rod are connected by nonhelicallinker (L1, L1-2, L2) regions. The amino acid (aa) regions in black barsrepresent 5 domains that were examined for K8/K18 mutations. Theremaining regions in gray bars contain 10 exonic K8/K18 domains thathave been analyzed for mutations. (B) PCR products, from a controlpatient (with K8 WT) and a patient with K8 R340H, were analyzed bydenaturing HPLC using a WAVE® System. The control (K8 WT; SEQ ID NO:5,SEQ ID NO:6) is characterized by one major peak, while the K8 R340H; SEQID NO:7, SEQ ID NO:8 shows a different chromatogram due to resolution ofthe homoduplexes from the heteroduplexes, thereby suggesting thepresence of a K8 mutation. Electropherograms from DNA sequencing confirmthe presence of a K8 R340H heterozygous missense mutation (CGT→CAT).

FIGS. 2A-2B: Protein expression of mutant K8 and K18 in explantedlivers. (A): K8/K18 immunoprecipitates were obtained from 1%Empigen-solubilized normal liver or livers with keratin mutation. Theimmunoprecipitates were separated by isoelectic focusing followed bySDS-PAGE, then immunoblotting with anti-K8/K18 antibodies. Note that K8and K18 in normal liver consist of two or three isoforms depending ontheir phosphorylation levels (a, b). In contrast, some of the mutantkeratins contain four (K8) or five (K18) isoforms due to coexpression ofthe wild-type and mutant keratin with subsequent generation of alteredcharged species that have a slightly different mutation-inducedisoelectric focusing point (d, f, g, h). (B): K8/K18 immunoprecipitateswere prepared from normal liver or liver with the K18 T102A mutation,then analyzed by SDS-PAGE. The K18 bands were cut out, digested withtrypsin, then analyzed with a MALDI-TOF mass spectrometer. Note that apeak position at 818.3 was detected only in the liver specimen with theK18 T102A mutation but not in normal liver. The mass difference of 30,between the wild-type and T102A K18 tryptic peptides (848.3 versus818.3), corresponds to the HO—C—H species (two hydrogen, one oxygen andone carbon atoms with a mass of 30 daltons) that are present inthreonine (the wild-type residue) but not in alanine (the mutantresidue).

FIGS. 3A-3C: K8 R340H mutation-proximal comparison of type II keratinsequences and confirmation of K8 R340H mutant protein expression inexplanted livers. (A) (SEQ ID NO:9) Single letter abbreviations are usedto represent amino acids. Bold dots represent amino acids that areidentical to the K8 sequence. The shaded area highlights the conservedR340 of K8 and shows the histidine mutation we identified. Note that theK8 R340-containing motif (AEQRG; SEQ ID NO:9, residues 4-8) is highlyconserved in type II keratins. It is also conserved across species,being found in mouse and frog K8. (B) BHK cells were transientlycotransfected with K8/K18 WT or K8 R340H/K18 WT. K8/K18immunoprecipitates were obtained from 1% NP40-solubilized cell lysates.The immunoprecipitates were analyzed by SDS-PAGE, followed byimmunoblotting with anti-K8 R340 or anti-K8 H340 epitope-specificantibodies that preferentially recognized K8 WT or K8 R340H mutant,respectively. (C) K8/K18 immunoprecipitates were obtained from 1%NP40-solubilized normal liver or livers with the K8 R340H mutation.Samples were analyzed as described in panel B. Note that anti-K8 R340(WT) recognizes K8 in the control patient (with K8 WT) and the patientwith K8 R340H (lanes 1-6), whereas anti-K8 H340 (mutant) recognizes onlypatient livers with the K8 R340H mutation (lanes 2-6) but not the normalliver (lane 1). This indicates that the patients with K8 R340H mutationare heterozygous with regard to the keratin mutation. Arrowheadscorrespond to degraded K8.

FIGS. 4A-4B: Keratin filament organization in human liver explants, andhistologic findings of livers harboring the keratin mutations. (A):Human livers were sectioned, fixed in acetone and double-stained withrabbit anti-K8/18 or mouse anti-vimentin antibodies. Inset in panel ishows control double staining using fluorochrome-conjugated goatanti-rabbit and goat anti-mouse antibodies without adding any primaryantibodies. All images were obtained using the same magnification. Barin panel a=20 μm. (B): Hematoxylin and eosin staining of explanted liverfrom two patients with acute fulminant hepatitis. Panel “a” is from apatient without a keratin mutation while panel “b” is from a patientwith the K18 T102A mutation. The region outlined by a box in “b” ismagnified in panel “c” to illustrate the cytoplasmic filamentousdeposits noted primarily in livers of patients with keratin mutations.

FIG. 5: Distribution of K8 and K18 mutations within the keratin proteinbackbone. The H1 and V1 subdomains of the K8 head domain, and the aminoacid positions of the K8 and K18 domains/subdomains are indicated. Forthe K8 head/tail and K18 head/rod domains, each arrowhead represents anindependently identified mutation at the indicated residue. The 30 K8R340H mutations are highlighted with a single large arrowhead. Note thatthe most common K8/K18 mutation to date is the newly identified K8 R340H(30 of 58, 6.4%), with the next most common mutations being K8 G61C (6of 58) and K8 Y53H (5 of 58). Three patients had a double mutant (K8R340H and K18 R260Q, K8 R340H and K18 T102A, K18 I149V and K18 T294M).Shaded areas correspond to “hot spots” of epidermal keratin mutations.Note the non-overlap of the location of K8/K18 mutations versus themajor location of epidermal keratin mutations.

FIG. 6: Schematic model of the effect of keratin mutations on filamentorganization and subsequent liver injury: Hepatocytes contain anextended array of filamentous keratin and a small soluble keratin pool.A variety of physiologic and nonphysiologic stimuli may result inreversible keratin filament reorganization. However, in the presence ofkeratin mutations, the keratin filaments may be unstable (as for the K8Y53H mutation upon heat stress or exposure to okadaic acid), or may notbe able to organize normally (as for the K8 G61C mutation upon oxidativestress; e.g. in the presence of H₂O₂). Filament instability and abnormalorganization may then predispose to liver injury via a variety ofmechanisms that include the role of keratins in apoptosis, cellsignaling or protein targeting to subcellular compartment.

The above six figures demonstrate: (i) the presence of the keratinmutations at the protein level (FIGS. 2 and 3), (ii) althoughimmunofluorescence staining is not significantly altered in the liverswith keratin mutation as compared to those without keratin mutation(FIG. 4A) there appears to be a unique histologic feature of cytoplasmicfilamentous deposits that are observed primarily in livers of patientswith keratin mutations, (iii) distribution of K8 and K18 mutantions inthe keratin protein backbone (FIG. 5) and (iv) schematic model of theeffect of keratin mutations on filament organization and subsequentliver injury (FIG. 6).

DETAILED DESCRIPTION OF THE EMBODIMENTS

Methods and compositions are provided for the diagnosis of apredisposition to liver or biliary tract disease, including, withoutlimitation, viral hepatitis, autoimmune liver disease, biliary atresia,alcoholic cirrhosis and other acute or chronic toxic liver injury,cryptogenic cirrhosis, acute fulminant hepatitis, primary sclerosingcholangitis, primary biliary cirrhosis, diseases that are linked withcryptogenic cirrhosis, such as nonalcoholic steatohepatitis, and thelike. The invention is based, in part, on the evaluation of the K8/K18keratin genotype, for which alleles predisposing to disease are hereinidentified. This permits the definition of disease pathways and theidentification of a target in the pathway that is useful diagnostically,in drug screening, and therapeutically.

In one aspect of the present invention, methods are provided fordetermining a predisposition to liver disease in an individual. Themethods comprise an analysis of genomic DNA in an individual for anallele of keratin K8 or K18 that confers an increased susceptibility toliver disease. Individuals are screened by analyzing their genomic K8and/or K18 gene sequence in blood or tissue/cell specimen for thepresence of a predisposing allele, as compared to a normal sequence.Screening for the presence of the mutation can also be done usingantibodies that specifically identify the keratin mutation, or screeningof genomic material in serum.

In addition to the provided sequence polymorphisms, the effect of acandidate polymorphism in a K8 or K18 sequence can be determined forassociation with a predisposition to liver disease. The candidatepolymorphism may be analyzed, for example, for segregation of thesequence polymorphism with the disease phenotype. A predisposingmutation will segregate with incidence of the disease. Alternatively,biochemical studies may be performed to determine whether a candidatepolymorphism affects the quantity or quality (in terms of itsdistribution, interaction with binding partners, etc), or function ofthe protein.

Intermediate filaments (IFs) are a structurally related family ofcellular proteins that are intimately involved with the cytoskeleton.The common structural motif shared by all IFs is a central alpha-helical‘rod domain’ flanked by variable N- and C-terminal domains. The roddomain, the canonical feature of IFs, has been highly conserved duringevolution. The variable terminals, however, have allowed the known IFsto be classified into 6 distinct types by virtue of their differingamino acid sequences. Keratins compose types I and II IFs. Type I andtype II keratins are usually expressed as preferential pairs, in equalproportions in cells, of type I and type II keratins.

Human keratin 18 is a type I IF protein subunit, whose expression isrestricted in adults to a variety of so-called “simple-type” epithelialtissues. KRT18 is highly divergent among the type I keratins withN-terminal and C-terminal domains that are quite different from those ofepidermal keratins. The KRT18 gene is 3,791 by long and the keratin 18protein is coded for by 7 exons. The genetic sequence of K18 may befound in Genbank, accession no. NM_(—)000224. The exon structure ofKRT18 has been conserved compared to that of other keratin genes, withthe exception of a single 3-prime terminal exon that codes for the taildomain of the protein that is represented by 2 exons in epidermalkeratins. Keratin 8 is a type II keratin, which is co-expressed withK18. The genetic sequence of K8 may be found in Genbank, accession no.NM_(—)002273.

Mutations in K8 that are associated with a predisposition to liverdisease (all the amino acid numbers represent amino acids of theprocessed protein) include G52V (GGC→GTC); Y53H (TAT→CAT); G61C(GGC→TGC); R340H (CGT→CAT); G433S (GGC→AGC); R453C (CGC→TGC); 1-465(I)RDT(468) (frameshift). The K8 frameshift mutation at Ile-465 generates atruncated 468 (instead of 482) amino acid protein that contains aminoacids 1-465, and three additional new amino acids (RDT) after theframeshift mutation. Mutations in K18 that are involved with apredisposition to liver disease include Δ64-71(TGIAGGLA) (deletion);T102A (ACC→GCC); H127L (CAT→CTT); 1149V (ATC→GTC); R260Q (CGG→CAG);E275G (GAG→GGG); Q284R (CAG→CGG); T294M (ACG→ATG); T2961 (ACA→ATA);G339R (GGG→AGG). The K18 N-terminal domain deletion (Δ64-71) generated a421 (instead of 429) amino acid protein (see Table 3).

Keratin K8 or K18 mutations that result in an amino acid substitution ordeletion at any one of the mutated positions designated above may begenerally defined to include K8 G52X; Y53X; G61X; R340X; G433X; R453Xand K18 T102X; H127X; I149X; R260X; E275X; Q284X; T294X; T296X; G339X,where X is any amino acid other than the naturally occurring amino acidas set forth in the published sequence of K8 or K18, and X may alsorefer to a deleted amino acid or amino acids. Mutations may also bespecified in terms of the DNA sequence, and could include any deletions(in addition to those described above), or mutations in the promoter orother regulatory regions that may affect RNA levels or stability underbasal conditions or in response to any type of mechanical ornonmechanical stress that the liver may be exposed to due to internal orexternal factors.

DNA encoding a K8/K18 protein may be cDNA or genomic DNA or a fragmentthereof that encompasses one or more of the above identifiedpolymorphisms. As known in the art, cDNA sequences have the arrangementof exons found in processed mRNA, forming a continuous open readingframe, while genomic sequences may have introns interrupting the openreading frame. The term K8/K18 gene shall be intended to mean the openreading frame encoding such specific polypeptides, as well as adjacent5′ and 3′ non-coding nucleotide sequences involved in the regulation ofexpression, up to about 1 kb beyond the coding region, in eitherdirection.

The nucleic acid compositions of the subject invention encode all, none,or a part of a K8 or K18 polypeptide comprising a polymorphism asdescribed above. Fragments may be obtained of the DNA sequence, or ofthe mRNA, by chemically synthesizing oligonucleotides in accordance withconventional methods, by restriction enzyme digestion, by PCRamplification, etc. For the most part, DNA fragments will be at leastabout 25 nt in length, usually at least about 30 nt, more usually atleast about 50 nt. For use in amplification reactions, such as PCR, apair of primers will be used. The exact composition of the primersequences is not critical to the invention, but for most applicationsthe primers will hybridize to the subject sequence under stringentconditions, as known in the art. It is preferable to chose a pair ofprimers that will generate an amplification product of at least about 50nt, preferably at least about 100 nt. Algorithms for the selection ofprimer sequences are generally known, and are available in commercialsoftware packages. Amplification primers hybridize to complementarystrands of DNA, and will prime towards each other.

The subject K8/K18 genes are isolated and obtained in substantialpurity, generally as other than an intact mammalian chromosome. Usually,the DNA will be obtained substantially free of other nucleic acidsequences that do not include a K8/K18 sequence or fragment thereof,generally being at least about 50%, usually at least about 90% pure andare typically “recombinant”, i.e. flanked by one or more nucleotideswith which it is not normally associated on a naturally occurringchromosome. The subject nucleic acids may be used to identify expressionof the gene in a biological specimen.

A number of methods may be used for determining the presence of apredisposing mutation in an individual. Genomic DNA may be isolated fromthe individual or individuals that are to be tested. DNA can be isolatedfrom any nucleated cellular source such as blood, hair shafts, saliva,mucous, biopsy, feces, etc. mRNA may also be reverse transcribeddepending on the detection method. Methods using PCR amplification canbe performed on the DNA from a single cell, although it is convenient touse at least about 10⁵ cells. Where large amounts of DNA are available,the genomic DNA is used directly. Alternatively, the region of interestis cloned into a suitable vector and grown in sufficient quantity foranalysis, or amplified by conventional techniques. Of particularinterest is the use of the polymerase chain reaction (PCR) to amplifythe DNA that lies between two specific primers. The use of thepolymerase chain reaction is described in Saiki et al. (1985) Science239:487, and a review of current techniques may be found in McPherson etal. (2000) PCR (Basics: From Background to Bench) Springer Verlag; ISBN:0387916008. A detectable label may be included in the amplificationreaction. Suitable labels include fluorochromes, e.g. fluoresceinisothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin,allophycocyanin, 6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

Primer pairs are selected from the K8/K18 genomic sequence usingconventional criteria for selection. The primers in a pair willhybridize to opposite strands, and will collectively flank the region ofinterest. The primers will hybridize to the complementary sequence understringent conditions, and will generally be at least about 16 nt inlength, and may be 20, 25 or 30 nucleotides in length. The primers willbe selected to amplify the specific region of the K8/K18 gene suspectedof containing the predisposing mutation. Multiplex amplification may beperformed in which several sets of primers are combined in the samereaction tube, in order to analyze multiple exons/introns/promoter andother regulatory regions simultaneously. Each primer may be conjugatedto a different label.

After amplification, the products may be size fractionated and evaluatedfor sequence polymorphisms. Fractionation may be performed by gelelectrophoresis, particularly denaturing acrylamide or agarose gels. Aconvenient system uses denaturing polyacrylamide gels in combinationwith an automated DNA sequencer, see Hunkapillar et al. (1991) Science254:59-74. The automated sequencer is particularly useful with multiplexamplification or pooled products of separate PCR reactions. Capillaryelectrophoresis may also be used for fractionation. A review ofcapillary electrophoresis may be found in Landers, et al. (1993)BioTechniques 14:98-111. Hybridization with the variant sequence mayalso be used to determine its presence, by Southern blots, dot blots,etc. Single strand conformational polymorphism (SSCP) analysis,denaturing gradient gel electrophoresis (DGGE), and heteroduplexanalysis in gel matrices is used to detect conformational changescreated by DNA sequence variation as alterations in electrophoreticmobility or via HPLC type analysis (eg WAVE® System). The hybridizationpattern of a control and variant sequence to an array of oligonucleotideprobes immobilized on a microarray, may also be used as a means ofdetecting the presence of variant sequences.

The presence of a predisposing mutation is indicative that an individualis at increased risk of developing liver disease. The diagnosis of adisease predisposition allows the affected individual to seek earlytreatment, dietary measures, to avoid activities that increase risk forliver disease, and the like.

In addition to genetic tests, the presence of the mutated polypeptidemay be detected, by determination of the presence of polypeptidecomprising the mutation using analytic methods such as mass spectrometryor immune-related methods such as mutant-specific antibodies, or bydetecting the presence of cytoplasmic filamentous deposits.

In a typical assay, a liver sample is assayed for the presence of K8and/or K18 specific sequences by combining the sample with a K8 and/orK18 specific binding member, and detecting directly or indirectly thepresence of the complex formed between the two members. The term“specific binding member” as used herein refers to a member of aspecific binding pair, i.e. two molecules where one of the moleculesthrough chemical or physical means specifically binds to the othermolecule. In this particular case one of the molecules is K8 and/or K18,where K8 and/or K18 is any protein substantially similar to the aminoacid sequence of the human polypeptide sequences of this family, asdescribed above, or a epitope containing fragment thereof, and furthercomprising a predisposing mutation. The complementary members of aspecific binding pair are sometimes referred to as a ligand andreceptor. Testing can also be done using any source of genomic materialfrom a given individual or any tissue that also expresses K8 and K18 (eggastric, small or large intestinal biopsy).

In the present specification and claims, the term “polypeptidefragments”, or variants thereof, denotes both short peptides with alength of at least two amino acid residues and at most 10 amino acidresidues, oligopeptides with a length of at least 11 amino acidresidues, 20 amino acid residues, 50 amino acid residues, and up toabout 100 amino acid residues; and longer peptides of greater than 100amino acid residues up to the complete length of the native polypeptide.

Polypeptides detected by the present methods include naturally occurringalpha and beta subunits, as well as variants that are encoded by DNAsequences that are substantially homologous to one or more of the DNAsequences specifically recited herein, for example variants having atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or99.5% sequence identity. The specific binding pairs may include analogs,derivatives and fragments of the original specific binding member. Forexample, an antibody directed to a protein antigen may also recognizepeptide fragments, chemically synthesized peptidomimetics, labeledprotein, derivatized protein, etc. so long as an epitope is present.

Immunological specific binding pairs include antigens and antigenspecific antibodies or T cell antigen receptors. Recombinant DNA methodsor peptide synthesis may be used to produce chimeric, truncated, orsingle chain analogs of either member of the binding pair, wherechimeric proteins may provide mixture(s) or fragment(s) thereof, or amixture of an antibody and other specific binding members. Antibodiesand T cell receptors may be monoclonal or polyclonal, and may beproduced by transgenic animals, immunized animals, immortalized human oranimal B-cells, cells transfected with DNA vectors encoding the antibodyor T cell receptor, etc. The details of the preparation of antibodiesand their suitability for use as specific binding members are well-knownto those skilled in the art.

Alternatively, monoclonal or polyclonal antibodies are raised to K8and/or K18 polypeptides comprising a predisposing mutation. Theantibodies may be produced in accordance with conventional ways,immunization of a mammalian host, e.g. mouse, rat, guinea pig, cat,rabbit, dog, etc., fusion of resulting splenocytes with a fusion partnerfor immortalization and screening for antibodies having the desiredaffinity to provide monoclonal antibodies having a particularspecificity. These antibodies can be used for affinity chromatography,ELISA, RIA, and the like. The antibodies may be labeled withradioisotopes, enzymes, fluorescers, chemiluminescers, or other label,which will allow for detection of complex formation between the labeledantibody and its complementary epitope. Generally the amount of bound K8and/or K18 detected will be compared to negative control samples fromnormal tissue or cells.

Screening assays identify compounds that modulate the expression orstructure of K8/K18. Such compounds may include, but are not limited topeptides, antibodies, or small organic or inorganic compounds. Methodsfor the identification of such compounds are described below.

Cell- and animal-based systems can act as models for liver disease andare useful in such drug screening. The animal- and cell-based models maybe used to identify drugs, pharmaceuticals, therapies and interventionsthat are effective in treating liver disease. In addition, such animalmodels may be used to determine the LD₅₀ and the ED₅₀ in animalsubjects, and such data can be used to determine the in vivo efficacy ofpotential liver disease treatments. Animal-based model systems of liverdisease may include, but are not limited to, non-recombinant andengineered transgenic animals. Animal models exhibiting liver diseasesymptoms may be engineered by utilizing, for example, K8 and/or K18 genesequences in conjunction with techniques for producing transgenicanimals that are well known to those of skill in the art. For example,target gene sequences may be introduced into, and knocked out oroverexpressed in the genome of the animal of interest. Animals of anyspecies, including, but not limited to, mice, rats, rabbits, guineapigs, pigs, micro-pigs, goats, and non-human primates, e.g., baboons,monkeys, and chimpanzees may be used to generate liver disease animalmodels.

Any technique known in the art may be used to introduce a target genetransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell56:313-321); electroporation of embryos (Lo, 1983, Mol. Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc.

Specific cell types within the animals may be analyzed and assayed forcellular phenotypes characteristic of liver disease. Further, suchcellular phenotypes may include a particular cell type's fingerprintpattern of expression as compared to known fingerprint expressionprofiles of the particular cell type in animals exhibiting liver diseasesymptoms.

Cells that contain and express K8/K18 can be utilized to identifycompounds that exhibit pharmacologic activity of interest, in theprevention of liver disease. Cells of a cell type known to be involvedin liver disease may be transfected with sequences capable of increasingor decreasing the amount of K8 and/or K18 gene expression within thecell. For example, K8/K18 gene sequences may be introduced into, andoverexpressed in, the genome of the cell of interest, or, if endogenoustarget gene sequences are present, they may be either overexpressed or,alternatively disrupted in order to underexpress or inactivate targetgene expression.

Transfection of target gene sequence nucleic acid may be accomplished byutilizing standard techniques. Transfected cells can be evaluated forthe presence of the recombinant K8/K18 gene sequences, for expressionand accumulation of K8/K18 gene mRNA, and for the presence ofrecombinant K8/K18 protein. Where a decrease in K8/K18 gene expressionis desired, standard techniques may be used to demonstrate whether adecrease in expression is achieved.

In vitro systems may be designed to identify compounds capable ofpreventing or treating liver disease. Such compounds may include, butare not limited to, peptides made of D- and/or L-configuration aminoacids, phosphopeptides, antibodies, and synthetic or natural smallorganic or inorganic molecules. For example, assays may be used toidentify compounds that improve liver function involves preparing areaction mixture of K8/K18 and a test compound under conditions and fora time sufficient to allow the two components to interact, and detectingthe resulting change in the polypeptide structure. Alternatively, asimple binding assay can be used as an initial screening method. Theseassays can be conducted in a variety of ways. For example, one method toconduct such an assay would involve anchoring K8/K18 protein or a testsubstance onto a solid phase and detecting complexes anchored on thesolid phase at the end of the reaction. In another embodiment of such amethod, the assay tests the presence of products modulated by K8/K18.

In a binding assay, the reaction can be performed on a solid phase or inliquid phase. In a solid phase assay, the nonimmobilized component isadded to the coated surface containing the anchored component. After thereaction is complete, unreacted components are removed under conditionssuch that any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the previously nonimmobilizedcomponent is pre-labeled, the detection of label immobilized on thesurface indicates that complexes were formed. Where the previouslynonimmobilized component is not pre-labeled, an indirect label can beused to detect complexes anchored on the surface; e.g., using a labeledantibody specific for the previously nonimmobilized component (theantibody, in turn, may be directly labeled or indirectly labeled with alabeled anti-Ig antibody).

Alternatively, a binding reaction can be conducted in a liquid phase,the reaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for target geneproduct or the test compound to anchor any complexes formed in solution,and a labeled antibody specific for the other component of the possiblecomplex to detect anchored complexes.

Cell-based systems such as those described above may be used to identifycompounds that act to ameliorate liver disease symptoms. For example,such cell systems may be exposed to a test compound at a sufficientconcentration and for a time sufficient to elicit such an ameliorationof liver disease symptoms in the exposed cells. After exposure, thecells are examined to determine whether one or more of the liver diseasecellular phenotypes has been altered to resemble a more normal or morewild type, non-liver disease phenotype. Liver disease “symptoms” incells include surrogate markers such as changes in keratin filamentorganization, keratin properties (such as phosphorylation orsolubility), or interaction with a binding partner.

In addition, animal-based disease systems, such as those described,above may be used to identify compounds capable of ameliorating diseasesymptoms. Such animal models may be used as test substrates for theidentification of drugs, pharmaceuticals, therapies, and interventions,which may be effective in treating or preventing disease. For example,animal models may be exposed to a compound, suspected of exhibiting anability to ameliorate liver disease symptoms, at a sufficientconcentration and for a time sufficient to elicit such an ameliorationof disease symptoms in the exposed animals. The response of the animalsto the exposure may be monitored by assessing the reversal of disordersassociated with disease.

With regard to intervention, any treatments that reverse any aspect ofliver disease symptoms may be considered as candidates for human diseasetherapeutic intervention. Dosages of test agents may be determined byderiving dose-response curves.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates may beformulated for administration by oral, buccal, parenteral or rectaladministration.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated to give controlled release ofthe active compound. For buccal administration the compositions may takethe form of tablets or lozenges formulated in conventional manner.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one specific K8/K18nucleic acid reagent described herein, which may be conveniently used,e.g., in clinical settings, for prognosis of patients susceptible toliver disease.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing the subject components ofthe invention that are described in the publications, which componentsmight be used in connection with the presently described invention.

EXPERIMENTAL Methods

Patients: We included for the analysis specimens of 467 explanted liversthat were obtained from the liver transplantation units at StanfordUniversity, the University of California San Francisco, and CaliforniaPacific Medical Center. The 467 liver samples were previously examinedfor K8/K18 mutations in 5 exonic regions (Ku et al, N. Engl. J. Med.344:1580-1587, 2001 and Ku et al, Proc. Natl. Acad. Sci. USA.100:6063-6068, 2003), that included the epidermal keratin domains wheremost of the mutations have been identified (Irvine & McLean, Br. J.Dermatol. 140:815-828, 1999). Subsequent to this, the frequency ofK8/K18 mutations were determined in the remaining 10 exons of K8/K18.Peripheral blood samples from 349 healthy volunteers were obtained fromthe Stanford Blood Bank and used for genomic DNA isolation. In additionand when available, blood samples were obtained from patients with theidentified keratin mutations and/or from their children or parents. Thepatients' sex and racial/ethnic background were determined frompatients' medical records. No information could be found on 15 patientsdue to lack of records or to retransplant. For the control samples,anonymous information regarding sex and race was provided by theStanford Blood Bank. The diagnoses were based on the United Network forOrgan Sharing transplant listing. Medical records of all patients with akeratin mutation were reviewed and the diagnosis was confirmed.

Histopathology and statistical analysis: Pathology slides from theexplanted livers with keratin mutations, and matched liver diseasecontrols, were reviewed by a single pathologist who did not know whichspecimens harbored the keratin mutations. Slides from the explantedlivers of all 17 patients with keratin mutations, as well asdisease-matched controls, were examined and scored for the presence orabsence of features including Mallory and acidophil bodies, cell size,ground glass cytoplasm, and dysplasia. Images of the hematoxylin andeosin stained liver sections were obtained using a Nikon Eclipse E1000microscope with a 40× objective. Data analysis was conducted using theFisher's exact test performed with Statistical Analyzing System software(SAS).

Molecular methods: Genomic DNA was prepared using a Dneasy tissue kit(QIAGEN Inc., Chatsworth, Calif.). Exonic regions (FIG. 1A) wereamplified by the polymerase chain reaction. The amplified products wereanalyzed for mutation using the WAVE® System (Transgenomic Inc, SanJose, Calif.), and any samples with a “shift” pattern suggestive of amutation were sequenced in the forward and reverse directions to confirmthe presence of a mutation (FIG. 1B).

Biochemical methods: Tissues were homogenized in phosphate bufferedsaline containing 1% n-dodecyl-N,N-dimethylglycine (Empigen BB,Calbiochem-Novabiochem, San Diego, Calif.), 5 mM EDTA, and proteaseinhibitors. Homogenized samples were solubilized for 30 minutes,pelleted then used for immunoprecipitation of K8/K18. Precipitates wereanalyzed by: (i) SDS polyacrylamide gel electrophoresis (PAGE), underreducing or non-reducing conditions, then Coomassie staining, (ii)SDS-PAGE then immunoblotting, or (iii) two-dimensional gels usingisoelectric focusing (horizontal direction) and SDS-PAGE (verticaldirection) then immunoblotting.

Mass spectrometry analysis: Separated K8 and K18 bands were cut out frompreparative gels, reduced with dithiothreitol, alkylated withiodoacetamide, and then digested with trypsin in 50 mM ammoniumbicarbonate (pH 7.8) using a standard in-gel-digestion procedure.Extracted K8 or K18 tryptic peptides were desalted using a C18 ZipTip(Millipore, Mass.) then eluted with 50% acetonitrile-0.1%trifluoroacetic acid (TFA). A 1 μl aliquot of the eluant was mixed withequal volume of matrix solution (saturated a-cyano-4-hydroxycinnamicacid in 0.1% TFA-50% acetonitrile in water) and analyzed by a MALDI-TOFmass spectrometer (Bruker Biflex III) equipped with a nitrogen 337 nmlaser. The mass spectra were acquired in the reflectron mode. Internalmass calibration was performed with two trypsin autodigested fragments(842.5 and 2211.1 Da). K8 tryptic peptides were also digested with CNBrand similarly analyzed.

Antibody generation: Two polyclonal antibodies were generated afterimmunizing rabbits with synthetic peptides containing wild type K8 R340(³³⁵ADAEQRGELAI) or mutant K8 H340 (³³⁵ADAEQHGELAI). As shown in FIG.2B, anti-K8 R340 or anti-K8 H340 antibody recognized predominantly K8 WTor the K8 R340H mutant, respectively, that were co-expressed intransfected BHK cells with wild type K18.

Immunofluorescence staining: Snap-frozen liver explants were embedded inoptimum cutting temperature compound, sectioned then fixed in acetone(−20° C., 10 min). Sections were double stained with antibodies directedto K8/K18 (13) or vimentin (NeoMarkers, Freemont, CA). Flourescenceimages were obtained using an MRC 1024ES confocal scanner (BioRad,Hercules, Calif.) coupled to a Nikon Eclipse TE300 microscope.

Results

Identification of K8 and K18 mutations: We tested DNA extracted fromliver explants or peripheral blood for the presence of K8 or K18mutations. Two cohorts were examined, whose demographics are summarizedin Table 1: a group of 467 patients with a variety of liver diseases,and a control group of 349 blood bank donors. The ethnic background ofthe two cohorts was generally similar except for a higher preponderanceof Caucasian patients in the control group.

The etiology of the liver diseases is broad (Table 2), most of which isnoncryptogenic (based on clinical criteria, 68 of the 467 patients wereclassified as having cryptogenic liver disease; Table 2). We includedthe control blood bank cohort in order to address which mutationsidentified in the liver disease cohort are likely to represent “true”mutations versus polymorphism variants found in the general population.We previously reported that 7 heterozyous missense mutations in 5 exonicregions of K8/K18 were identified in 17 of 467 liver disease patientsand 2 of 349 blood bank controls (3.6% and 0.6% frequency; p<0.004) (Kuet al, Proc Natl Acad Sci USA. 100:6063-6068, 2003). The 5 exonicregions included the epidermal keratin domains where most of themutations have been identified in skin disease patients.

More recently, we analyzed the 10 remaining exonic regions for K8/K18mutations that were not examined previously (manuscript in preparation).Aside from several new polymorphisms, we identified 10 novel K8/K18amino acid heterozygous mutations (four K8 and six for K18) caused bydeletion (1), frameshift (1) and missense alterations (8). The K8frameshift mutation at Ile-465 generated a truncated 468 (instead of482) amino acid protein. The K18 N-terminal domain deletion (Δ64-71)generated a 421 (instead of 429) amino acid protein. The new K8/K18mutations were found in 44 of 467 patients and 11 of 349 controls (Table3). Three patients had a double mutant. One patient with primary biliarycirrhosis had K8 R340H and K18 R260Q, one patient with acute fulminanthepatitis had K8 R340H and K18 T102A, and one patient with unknown liverdisease etiology had K18 I149V and K18 T294M. Combining this recent datawith our previous data of all the patients identified with keratinmutations, the most common mutation is K8 R340H (30 of 58, ˜52%).

The mutations that were identified in 58 of 467 patients represent amutation frequency of 12.4%, as compared with a mutation frequency of3.7% found in 13 of 349 controls (P<0.0001; Tables 3 and 4). Given thedemographics of patients with keratin mutations, there does not appearto be any obvious accumulation of keratin mutations in a particular sexor ethnic background (Table 4). Since most of the specimen that weanalyzed consisted of liver explants, we also tested blood specimen forthe germline presence and transmission of the identified keratinmutations, in order to exclude the possibility that the mutations weidentified occurred during development of disease. Of the 58 independentpatients with K8/K18 mutations we were able to locate 4 patients and/ortheir offspring. All four of these patients and/or their offspring bloodspecimen had the identical heterozygous keratin mutation to thatidentified in the diseased explanted liver (Table 5). Therefore, theK8/K18 mutations we identified are not a consequence of the liverdisease but, rather, predispose their carriers to subsequent developmentof liver disease.

Expression of mutant keratin proteins: We examined, biochemically, theliver explant specimens from some of the mutation carriers within thecohort of 467 patients in order to confirm the presence of the mutationat the protein level. Mutations that were examined included 8 types: K8G52V/Y53H/G61C/R340H and K18 T102A/H127L/R260Q/G339R (Table 3). Thepresence of the K8 G61C protein was confirmed as we did previously (Kuet al, N Eng J Med 344:1580-1587, 2001), by formation of K8 dimers(under nonreducing gel conditions) due to the newly introduced cysteine(normally absent in K8/K18). We also used two-dimensional gels toseparate proteins based on their charge, and thereby confirmed thepresence of the K8 Y53H and K18 H127L/R260Q/G339R species (FIG. 2A) (Kuet al, Proc Natl Acad Sci USA 100:6063-6068, 2003). These four K8/K18mutations generated proteins with a different charge as compared totheir wild-type counterparts, and resulted in new isoforms (4 isoformsinstead of 2 for mutant K8, and 5 isoforms instead of 3 for mutant K18,FIG. 2A).

Two-dimensional gel analysis was, however, not informative for mutationsthat do not significantly alter the isoelectric point (FIG. 2A, K8 G52Vand K18 T102A variants). For these mutations, we compared the massspectrometric profiles of protease-generated fragments of wildtype andmutant keratins and tested for the presence of peptides that have amutation-altered mass (Ku et al, Proc Natl Acad Sci USA 100:6063-6068,2003). As shown in FIG. 2B, presence of the K18 T102A protein wasconfirmed by detection of its alanine-102-containing peptide with apredicted mass of 818.3 (in addition to the wild-typethreonine-102-containing peptide with a predicted mass of 848.3). Asimilar analysis was attempted for K8 G52V but no peptide thatcorresponds to a valine-to-glycine substitution was detected, likely dueto inability to recover it from the isolation column.

Given that the K8 R340H mutation was the most common mutation, found in30 of 58 affected individuals at the highly conserved K8 R340 (FIG. 3A),we generated and used polyclonal antibodies to examine the presence ofwild type K8 versus the mutant K8 R340H protein in patient liverexplants. The two antibodies we generated were directed towards the wildtype K8 R340 residue (³³⁵ADAEQRGELAI) or the mutant K8 H340 residue(³³⁵ADAEQHGELAI). Specificity of the antibodies was tested intransfected cells that expressed K8 WT or K8 R340H. Anti-K8 R340 (i.e.K8 WT) or anti-K8 H340 antibody recognized predominantly K8 WT or the K8R340H mutant, respectively (FIG. 3B). Expression of the K8 R340H proteinin patient liver explants was confirmed by immunoblotting of totalexplant liver lysates with the antibodies. As shown in FIG. 3C, anti-K8R340 (WT) recognizes K8 in the control patient and the patients with K8R340H (lanes 1-6), whereas anti-K8 H340 (mutant) recognizes only patientlivers with the K8 R340H mutation (lanes 2-6) but not the normal liver(lane 1). This indicates that the patients with K8 R340H mutation areheterozygous.

Effect of keratin mutations on keratin filament organization and liverhistology: We compared keratin filament organization in liver explantsof patients with and without keratin mutations (Ku et al, Proc Natl AcadSci USA 100:6063-6068, 2003). We did not observe any generalized keratinmutation-specific organization defects as tested by immunofluorescencestaining (FIG. 4A). The diseased livers (with or without keratinmutations) had reorganization of the keratins filaments with the mostprominent feature being thickening and partial collapse (FIG. 4A, panelsd and g) as compared with normal liver keratin staining (FIG. 4A, panela). The diseased livers had variable but significant vimentin-positivestaining (FIG. 4A, panels e, h), which was used as a fibroblast/stellatecell marker, as compared with normal liver (FIG. 4A, panel b). Vimentinstaining did not correlate with the presence of keratin mutations, anddid not involve hepatocytes (FIG. 4A; merged images c, f, i). Analysisof additional liver samples with proper attention to sample handlingwill be needed to better assess any potential keratin mutation-inducedeffects on keratin organization.

We also asked if any histologic features identified by light microscopycould distinguish cirrhotic livers of patients with and without keratinmutations (Ku et al, Proc Natl Acad Sci USA 100:6063-6068, 2003).Features such as Mallory's hyaline, acidophil bodies, enlargedhepatocytes, ground glass cytoplasm and dysplasia were found inkeratin-mutant and non-keratin-mutant livers. However, close inspectionof the liver specimen(s) showed a unique accumulation in somehepatocytes of cytoplasmic filamentous arrays (FIG. 4B). When coding ofthe slides (with mutant or nonmutant keratin) was opened, the findingsindicated that the filamentous deposits were found in 10 of 17 testedpatients with keratin mutations but in only 3 of 16 disease-matchedcontrols (p=0.03). When patients with only primary hepatocellulardiseases (cryptogenic, viral hepatitis, alcohol, acute fulminanthepatitis) were included in the analysis, 10 of 11 explants with akeratin mutation contained the cytoplasmic filamentous deposits ascompared with 3 of 13 disease-matched controls (p=0.001). The nature ofthe filamentous deposits remains to be determined, but they do notappear to correspond to aggregated keratins since they were notrecognized by anti-keratin antibodies (which may reflect epitopemasking, not shown).

Discussion

Mutations in keratins and other IF family members, including lamins,desmin, glial fibrillary acidic protein (GFAP) and neurofilaments, arewell-established causes of a wide range of tissue specific humandiseases. The list of newly identified diseases associated with IFproteins continues to grow, including the latest association of K8 withliver disease, GFAP with Alexander disease and the neurofilament-L chainwith Charcot-Marie-Tooth type-2. One distinguishing feature of K8/K18mutations, as compared with epidermal keratin mutations involvingK5/K14/K1/K10, is that the epidermal keratin diseases are typicallyautosomal dominant with ˜100% penetrance. In contrast, K8/K18 mutationsappear to be risk factors with variable penetrance, rather than directcauses of disease. In support of this, the location of the characterizedK8/K18 mutations does not involve conserved pan-keratin domain mutationhot spots that have been identified in epidermal keratins (FIG. 5).Apparent absence of such mutations suggests that they may be lethal,given that K8/K18 are among the earliest expressed keratins duringembryogenesis. Also, K8/K18 mutations are germline and do not simplyresult as a consequence of the liver disease (Table 5).

The ages of the offspring with the keratin mutations range from 31-52,but none of these carriers have apparent liver disease, based onclinical history and serologic testing. These observations support a“multi-hit” hypothesis, whereby one major “hit” is carrying a relevantK8/K18 mutation with subsequent “hits” including underlying liverdisease or exposure to injurious factors such as toxins or viruses (FIG.6). Clinical and natural history studies can be used to define therelative risk of subsequent development of liver disease, or therelative increase in progression of an underlying liver disease, inthose who carry specific K8/K18 mutations.

At the molecular level mutations in IF chains can, in principle, alterthe α-helical propensity of the chains, the number and type of theintra- and inter-helical ionic interactions, increase or decrease thestability of the α-helical strands, modify the helix capping potentialand change the hydrogen bonding ability of the chains. Mutations couldalso adversely affect the ability of the molecules to assemble in toviable IF, to bundle as normally required, or to function properly evenif assembled correctly (Table 6). Therefore, potential effects of thekeratin mutations include destabilizing K8/K18 filaments, interruptingionic interactions, introducing disulfide bonds, or altering keratinphosphorylation/solubility. These molecular consequences of keratinmutations may interfere the normal filament reorganizations that occurin hepatocytes upon multiple physiologic and nonphysiologic stimuli, andultimately result in liver disease (FIG. 6).

The significant number of patients described in this study, with new andpreviously described K8/K18 mutations, provide several insights intokeratin-associated liver diseases. For example, K8 Y53H, K8 G61C, andmost prominently K8 R340H are emerging as mutation hot spots. Thesemutations were found in 5, 6 and 30 of the 58 patients with K8/K18mutations, respectively (Table 3 and FIG. 5), and collectively make up˜71% of the K8/K18 mutations identified to date, while the K8 R340Hmutation alone makes up ˜52% of all K8/K18 mutations identified to date.Analysis of additional liver disease patients will help determine ifthese mutation hot spots maintain their frequency. Also, search foradditional keratin mutation carriers in a broad range of cryptogenic andnoncryptogenic liver diseases is clearly warranted.

When we initially identified K8 and K18 mutations in 6 patients, all 6had cryptogenic liver disease (Ku et al, J Clin Invest 99:19-23, 1997and Ku et al, N Engl J Med 344:1580-1587, 2001). This liver diseasemakes up nearly 10% of all patients who undergo liver transplantation.In this cryptogenic liver disease cohort 7 of 68 patients (10.3%) haveK8 or K18 mutations. However and more importantly, our more significantfindings are that keratin mutations are also common (12.8%) in thenoncryptogeic liver disease group (Table 2) that accounts for theremaining 90% of liver transplantations (based on the patient groups westudied). The association of keratin mutations with cryptogenic liverdisease raises the possibility that some diseases that are linked withthis type of cirrhosis, such as nonalcoholic steatohepatitis, may alsobe associated with keratin mutations. More K8 and/or K18 mutations maybe identified as we are in the process of completing thecharacterization of the entire coding and promoter regions (nearly 95%of K8 and K18 coding regions have been completed).

Although the mechanisms by which keratin mutations predispose tocirrhosis remain to be defined, already known and emerging keratinfunctions are likely to be involved. For example, multiple transgenicmouse model studies showed that K8/K18 serve the essential function ofprotecting hepatocytes from a variety of stresses including agents thatcause acute (e.g. acetaminophen) or chronic (e.g. griseofulvin) injury,and agents that induce apoptosis (e.g. Fas antibody). K8/K18 may also beinvolved in protein targeting to the apical compartment of polarizedepithelia, interacting with apoptotic machinery proteins, cell signalingand regulating the availability of abundant cellular proteins. Hence,keratin mutations may potentially act at a number of functional cellularnodes. One surrogate marker of keratin function is cytoplasmic filamentorganization, which was shown to be abnormally altered, only afterstress exposure, in the K8 Y53H/G61C mutations (Ku et al, N Engl J Med344:1580-1587, 2001). Our observation of preferential cytoplasmicfilamentous deposits in cirrhotic livers of patients with keratinmutations is likely to be relevant and is reminiscent of Rosenthalfibers that are seen in association with Alexander Disease. The natureand pathogenesis of these deposits and their association withkeratin-related liver disease remain to be investigated, but they aremorphologically distinct from Mallory body-type deposits.

It is evident that subject invention provides a convenient and effectiveway of determining whether a patient will be susceptible to liverdisease. The subject methods will provide a number of benefits,including preventive treatment and diet. As such, the subject inventionrepresents a significant contribution to the art.

1. A method for detecting a predisposition to liver disease in anindividual, the method comprising: analyzing an individual forquantitative or qualitative change in phenotype or genotype of keratinK8 or K18.
 2. The method of claim 1, wherein said liver disease is anoncryptogenic liver disease.
 3. The method of claim 2, wherein saidhuman keratin genotype is one or more of K8 G52X; Y53X; G61X; R340X;G433X; R453X and K18 T102X; H127X; I1149X; R260X; E275X; Q284X; T294X;T296X; G339X, where X is any amino acid other than the naturallyoccurring amino acid or a deleted amino acid. The mutant genotype mayalso involve other mutation sites, or deletion regions, that involve thecoding or noncoding regions of the K8 or K18 genes.
 4. The method ofclaim 3, wherein said analyzing the genomic or mRNA sequences comprisesthe steps of: amplifying a region of the K8 or K18 coding or noncodingsequences from isolated genomic DNA or mRNA to provide an amplifiedfragment; detecting the presence of a polymorphic sequence in saidamplified fragment.
 5. The method of claim 4, wherein said detectingstep comprises hybridization with a probe specific for the sequence ofsaid polymorphism.
 6. The method of claim 3, wherein said detecting stepcomprising contacting a cell, tissue or potentially a serum sample withan antibody specific for one or more of said polymorphisms.
 7. A methodof screening for biologically active agents that affect susceptibilityto liver disease, the method comprising: combining a candidatebiologically active agent with any one of: (a) a K8/K18 polypeptidecomprising one or more of K8 G52X; Y53X; G61X; R340X; G433X; R453X andK18 T102X; H127X; I149X; R260X; E275X; Q284X; T294X; T296X; G339X, whereX is any amino acid other than the naturally occurring amino acid or adeleted amino acid; the polypeptides may also comprise deletions in K8and/or K18; (b) a cell comprising a nucleic acid encoding a K8/K18polypeptide comprising one or more of K8 G52X; Y53X; G61X; R340X; G433X;R453X and K18 T102X; H127X; I149X; R260X; E275X; Q284X; T294X; T296X;G339X, where X is any amino acid other than the naturally occurringamino acid or a deleted amino acid; or a cell that expresses a deletionof K8 and/or K18; or a cell expressing another K8 or K18 mutant thatalters K8/K18 filament organization such as the K18 R89c which causeskeratin filament collapse; or (c) a non-human transgenic animal modelfor liver disease comprising an exogenous and stably transmitted geneencoding a K8/K18 polypeptide comprising one or more of K8 G52X; Y53X;G61X; R340X; G433X; R453X and K18 T102X; H127X; I149X; R260X; E275X;Q284X; T294X; T296X; G339X, where X is any amino acid other than thenaturally occurring amino acid or a deleted amino acid; or a transgenicanimal model expressing a deletion of K8 and/or K18; or a transgenicanimal model expressing another K8 or K18 mutant that alters K8/K18filament organization such as the K18 R89c which causes keratin filamentcollapse and determining the effect of said agent susceptibility toliver disease.